Ischemic brain injury is a common disorder among veterans, but the underlying mechanisms of it are not completely understood. Our latest studies strongly suggest that abnormal protein aggregation and multi-organelle damage lead to delayed neuronal death after brain ischemia. There are two major routes for clearance of protein aggregates and damaged organelles: (i) the ubiquitin-proteasomal system; and (ii) the autophagy pathway. Very recently, a renaissance in the autophagy field has shed light on many areas of biological diseases. The autophagy pathway is the chief route for bulk degradation of protein aggregates and aberrant organelles. Failure of autophagy leads to accumulation of protein aggregates and aberrant organelles, resulting in delayed neuronal death. The objective of this proposal is to study the impairment of autophagy after brain ischemia. The hypothesis is that brain ischemia leads to disruption of the autophagy pathway via irreversible inactivation of N-ethylmaleimide-sensitive fusion protein (NSF) ATPase, resulting in multiple organelle damage/failure and delayed neuronal death. Our latest studies show that accumulation of: (i) autophagy vacuoles (AVs), (ii) protein aggregates, and (iii) aberrant organelles are the most prominent early ultrastructural changes in neurons after brain ischemia. These new results clearly indicate that the autophagy pathway is severely damaged after brain ischemia. Our studies further show that impairment of the autophagy pathway is mainly attributable to irreversible inactivation of NSF after brain ischemia. NSF is the key ATPase for AV-to-lysosome fusion, and is completely inactivated in neurons undergoing delayed neuronal death after brain ischemia. We therefore generated an NSF- deficient transgenic mouse line. The dominant pathologic phenotype of this transgenic mouse line is continuous buildup of AVs and damaged organelles, which is followed by delayed neuronal death, virtually replicating the pathological changes observed after brain ischemia. Aim 1 will study the mechanisms of autophagy impairment after brain ischemia by analyzing all key autophagy and NSF-related proteins. We will investigate: (i) whether the autophagy pathway fails to keep up with the generation of damaged organelles after brain ischemia; (ii) if this failure is due to malfunction of the NSF-dependent AV-to-lysosome fusion; (iii) whether NSF inactivation contributes to selective neuronal vulnerability; and (iv) if presynaptic NSF-related presynaptic proteins also contribute to ischemic neuronal injury. Aim 2 will investigate the specific role of NSF in the impairment of the autophagy pathway after brain ischemia by using inducible and neuron-specific NSF loss/gain-of-function mouse models. This aim will test the prediction that NSF deficiency will disrupt the autophagy pathway, leading to delayed neuronal death, whereas overexpression of functional NSF will restore autophagy deficiency and offer neuroprotection after brain ischemia. Aim 3 will explore: (i) ischemia-induced dysfunction of the autophagic clearance of damaged mitochondria; and (ii) if an increase in autophagic load by mtDNA damage leads to more severe ischemic neuronal injury. We will test this hypothesis in a quantitative manner using an inducible and neuron-specific mitochondrial DNA damage mouse model. This multi-approach proposal should provide key information about the mechanisms underlying the autophagy impairment and new strategies for treatment of ischemic brain injury.